INTRODUCTION:

The myriad diversity of plants and its associated natural compounds with immense pharmacological properties makes their study indispensible in the field of science. The plant natural products are known to show various properties like anti-inflammatory, antiviral, antitumor, antimalarial, and analgesic etc and have revolutionized the medical industry. Statistics reveal that contribution of phytomedicines in health accounts to 65 % in Indian population, 80% in African population and according to WHO approximately 80 % of the world population depends on plant based medicines¹. The drug design process based on pharmacologically important compounds follows three approaches. Firstly, rational drug design where drugs are tailor-made for the targets.

Secondly, combinatorial chemistry where in large number of compounds are screened for a target. And lastly, isolation of new bioactive compounds from plants. The first two methods are preferred by the pharmaceutical industry as they involve advanced techniques that would cut the time of developing a drug. But these methods are costly and would be successful if the disease mechanism is very vivid which is not always possible. They being synthetic are perceived as unhealthy on a long term basis. Hence now focus is applied on the re-searching and re-exploring the enormous natural plant resource for natural compounds as leads. The plant based molecules are diverse in their structure and function, as they developed out of evolutionary stress there by making them appropriate as lead molecules in drug discovery process². The choice of plants for the study is well supported by the ethno botanical facts of its medicinal properties. This paved way for the scientists to bioprospect plants for novel compounds with biological activities. Though isolating new compounds from plants is time consuming, the improvements in the analytical techniques like mass spectrometry, high-performance liquid chromatography and higher magnetic field-strength nuclear magnetic resonance instruments made the study more feasible and interesting. These advances are increasing the repository of novel natural compounds.

Apart from being an immense source of natural compounds, plants are inhabited internally by diverse microbial communities comprising bacterial, archaeal, fungal, and protistic taxa. These microorganisms showing endophytic lifestyles play crucial roles in plant development, growth, fitness, and diversification. Additionally, they also are extensive producers of assorted secondary metabolites whose function ranges from anticancer, antioxidant, antidiabetic, immunosuppressive, antifungal, anti-oomycete, antibacterial, insecticidal, nematicidal to antiviral agents^3,4,5.

In this context, an attempt has been made to study the medicinal plant Bauhinia purpurea popularly known in Ayurveda to pacify all the three doshas and is beneficial in diseases like Chronic lymphadenitis (Apaci), Goiter (Gandamala), Worm infestation (Krmiroga), Wound (Vrana), Prolapsed rectum (Gudabhramsa).

MATERIAL AND METHODS:

Selection of plant:

The plant that is selected for present study is Bauhinia purpurea due to its various benefits. The plant is reported to show activities like antibacterial, anthelmintic, analgesic, antidiabetic, laxative, etc. It also has many cures like abscess, convulsions, cough, flatulence, obesity, leprosy, rheumatism, jaundice, piles, ulcer, wounds, etc. It contains notable compounds like L-Dopa, astragalin, beta sitosterol, etc. Almost the entire plant is used in the healing process like its bark, root, leaf, stem and flower. Predominantly the bark extract is used as it is reported to increase the production of both T3 and T4 levels⁶

Collection of plant material:

The fresh and healthy stems, leaves and flowers of Bauhinia purpurea plant were collected from Chennai district, Tamilnadu.

Extract preparation for phytochemical analysis:

The first step involved in extract preparation is to dry and grind the plant material. The collected specimens (stem, leaf and flower) of Bauhinia purpurea were initially washed with running tap water to remove excess debris and shade dried for 6-10 days. Protection from direct sunlight is advised to minimize chemical reactions induced by UV rays. The next step is to grind the sample into finer particles in size. The basic principle behind grinding is to increase the surface area for extraction thereby increasing the rate of extraction. Mechanical (mixer) grinder was used to grind the sample well. In order to aid decrease in sample’s particle size (tea) filter was used. The powdered sample was stored in air tight containers for the phytochemical analysis. About 5 gm of the dried sample powder (mixture of stem, leaf and flower) was extracted with the solvents of increasing polarity from a nonpolar to more polar solvents such as chloroform, ethyl acetate, ethanol and water to ensure that a wide polarity range of compounds could be extracted by the conventional serial exhaustive method. Sample was soaked with each solvent for about 24 hours at a room temperature (solvent to sample dry weight ratio of 10:1 (v/w))⁷. Each time before extracting with next solvent, the powdered material was air dried below 50˚C and then subjected to further extraction. The collected extracts were subjected to phytochemical screening for the identification of the different phytocompounds.

Determination of extractive yield:

The extractive values of the powder of Bauhinia purpurea are determined with different solvents i.e. Ethyl acetate, chloroform, ethanol and water⁸. The percentage extractive yield was calculated by formula as mentioned below,

% Extractive yield (w/w) = Weight of dried extract (g) / Weight of dried sample (g) × 100

Phytochemical analysis:

The different extracts of B. purpurea were subjected to phytochemical analysis, a preliminary qualitative phytochemical screening technique to determine the presence of various plant metabolites according to the procedure of Horbone et al and Kokate et al^9,10.

Characterization of phytocompounds:

Thin Layer Chromatography (TLC):

TLC is a chromatographic technique that is used to separate mixture of compounds on the basis of their partition coefficient. It is a very commonly used technique in synthetic chemistry for identifying compounds, determining their purity and following the progress of a reaction. In the present study three solvent systems are used in order to separate the components mixture in each of the four extracts used namely chloroform, ethyl acetate, ethanol and water. The three solvent systems are: 1. Chloroform 2. Ethyl acetate: n- hexane (6:4) 3. n-hexane: Ethyl acetate: methanol: chloroform (4:4:2:1). TLC techniques have been developed and successfully applied for qualitative analysis of antioxidants.

Screening of antioxidant activity by Bioautography:

2,2-diphenyl-1-picrylhydrazylradical (DPPH) has an absorption maximum at 517nm, which decreases upon reduction through reaction with a radical scavenger. The corresponding color change can thus be observed in a TLC bioassay. The developed chromatogram is sprayed with a solution of 0.2% DPPH in methanol/ethanol. The plate is examined in daylight after 30 min. Free radical scavengers appear as cream/yellow spots against a purple back ground¹¹.

High Performance Liquid Chromatography (HPLC):

The methanolic extract of B. purpurea was performed using 515 HPLC pumps and 2489 UV/VIS detectors of Waters company, USA, having reverse phase water guard Column: Symmetry C18 (5μm, 4.6*250mm) and Hamilton microliter syringe using an injection volume of 20 μl. The data analysis is done using Empower 2 software. In general, it was found that UV detection was quite pronounced at wavelengths of 280 nm and 360 nm using a flow rate of 1.0 ml/min. The mobile phase gradient elutions and run times used in the method is

1. Total run time is 25 minutes

2. Gradient elution of two solvents is used- Solvent A (Methanol) and Solvent B (1% Acetic acid in water). The gradient program is started with 60 % of A and then changed to obtain 35, 10, 60 and 60 % of eluent A at 5, 10, 15 and 20 minutes respectively.

Gas Chromatography- Mass Spectrometry (GCMS):

The phytochemical investigation of methanolic extract of B. purpurea is performed on a GC-MS equipment (Thermo Scientific Co.) Thermo GC-TRACE ultra ver.: 2.2, Thermo TSQ QUANTUM XLS Experimental conditions of GC-MS system were as follows: DB 5-MS capillary standard non-polar column, dimension: 30Mts, ID: 0.25 mm, Film thickness: 0.25μm. Flow rate of mobile phase (carrier gas: He) was set at 1.0 ml/min. In the gas chromatography part, temperature program (oven temperature) was 40°C raised to 290 °C at 5 °C/min and injection volume was 1.0 μl. A scan interval of 0.5 seconds with scan range of 40-600 m/z. Total GC running time was 35 min and the results were compared by using Wiley Spectral library search program.

Antibacterial activity of the plant extract:

Mueller Hinton agar plates are swabbed with 24 hrs old culture of Pseudomonas aeruginosa bacterial strain (it is a common type of bacteria that causes deadly disease in humans like joint infections, respiratory tract infections, etc.). Totally 6 wells are made in four different plates using sterile cork borer. Stock solutions of each plant extract namely chloroform, ethyl acetate, ethanol and water are prepared at a concentration of 1 mg/ml. About 20, 40, 60, 80 and 100µl of each plant extracts were added using sterile micropipette into the wells of each plate. Ciprofloxacin is used as a positive control. Later the plates are incubated at 37⁰C for 18-24 hours. After incubation the diameter of the inhibition zone (in mm) is measured. The experiment is carried in triplicates.

Antioxidant activity of the plant extract:

The DPPH method is used for estimating Free Radical Scavenging activity of the Chloroform extract¹² (Hatano et al 1988). 2 ml of methanolic solution of DPPH (0.1 mM) is mixed with various doses of 20-80μl of methanolic extract (4 mg/ml) in test tube and final volume of 3 ml is made with methanol. The absorbance of the mixture is measured after 40 min at 517 nm against methanol as blank. Ascorbic acid is used as standard and the experiments are carried out in triplicates. Scavenging activity is calculated using the formula:

% Scavenging activity = [(Ac-At)/Ac-As]x 100, where, Ac =absorbance of control, As = absorbance of standard and At = absorbance of test.

Isolation of endophytes:

Isolation of endophytes is based on the procedure described elsewhere¹³ (Sunkar and Nachiyar 2012) with slight modifications. The samples of B. purpurea (leaf, stem and flower) were rinsed gently in running tap water to remove dust and other debris. The plant material is cut into small pieces (0.5-1cm in length) in a sterile petriplate using forceps and scissors under laminar hood to ensure aseptic condition. The cut plant material is subjected to surface sterilization technique using 75% Ethanol (30s-1min), Sodium hypochlorite (2min) 75% Ethanol (15-30 sec) and Double distilled water (30 sec). These plant segments are now placed in nutrient agar and incubated for 24-72 hrs at 32°C for the growth of the bacteria. The endophytic bacteria growing out of the plant explants are sub cultured periodically on separate nutrient agar plates at room temperature and stored at 4˚C for further experiments.

Identification of endophytes:

Identification of endophytic bacteria is by two standardized techniques, namely biochemical test and 16S rRNA sequencing. The former represents the preliminary identification like which genus the bacteria belongs to and the latter gives complete information including its phylogenetic relationship.

Antibacterial activity and antioxidant activity of the endophytes:

The procedure employed to carry out the assay is provided above. The endophytic bacteria are cultured in nutrient broth for 24 hrs at 37°C. After incubation, the supernatant is separated by centrifugation and used as the sample for antibacterial activity and antioxidant activity.

RESULTS AND DISCUSSION:

Studies on exploring the biological potential of medicinal plants is on the rise as they are a part of traditional medicine since ages. This instigated us to explore the potential Bauhinia purpurea by analysing its phytochemicals and determining their biological activities. These plants are a store house of various microbes that dwell inside them and share a mutualistic relationship to sometimes pathogenic. These microbes known as endophytes are known to stock innumerable secondary metabolites of medical and industrial importance. Hence we tried to isolate endophytic bacteria from Bauhinia purpurea and characterise them. Reports exist on the studies with respect to Bauhinia purpurea but the studies were carried out using leaf extract and bark extract separately. We focused on checking the activities of solvent extracts of the mixture of plant parts that included stem, leaf and flower^14,15

Extraction and characterization of phytochemicals of Bauhinia purpurea:

Bauhinia purpurea is a medicinal plant that has been used from time immemorial by ancient Indians and Chinese for its tremendous medicinal wonders. In this study, from the freshly collected plant samples, the extracts were prepared by the method of serial exhaustive extraction. The physical parameters and extractive yield of different extracts was calculated by the formula mentioned earlier in methodology and tabulated below in table 1.

It can be inferred from the above table that the yield was higher for ethanolic extract (i.e.) 10.4 than that of other extracts which were 9, 8.5 and 6.2 for ethyl acetate, water and chloroform extracts respectively. Fig 1 represents the colour of each extract obtained from the extraction method mentioned earlier along with its characteristic appearance under florescence.

Fig 1 a) Chloroform Extract a’) Chloroform Extract under UV b) Ethyl acetate extract b’) Ethyl acetate extract under UV c) Ethanol Extract c’) c) Ethanol Extract under UV d) Water extract d’) Water extract under UV

Fluorescence analysis was performed to observe the variation in the colour of the extracts under UV light from that of the visible light. The results of fluorescence analysis showed pink colour for chloroform, ethyl acetate and ethanolic extracts which were dark green, green and light green under visible light. Water extract which was tawny brown under visible light appeared green in colour under UV light.

Phytochemical screening:

The presence of phytochemicals in Bauhinia purpurea was checked and the results were provided in the Table 2. The preliminary phytochemical tests were helpful in finding out the biochemical constituents present in the plant material that may be a source of pharmacologically active compounds.

Table 2 Phytochemical screening of different plant extracts

Sl. No	Test	Extracts
Sl. No	Test	Ethyl Acetate	Chloroform	Ethanol	Water
1.	Alkaloids	-	+	-	+
2.	Anthocyanin	+	-	-	-
3.	Saponin	-	-	+	+
4.	Triterpenoids	-	-	-	+
5.	Glycosides	-	-	+	+
6.	Theobromine	-	-	+	-
7.	Phenols	-	+	-	-
8.	Tannins	-	-	-	+
9.	Flavanoids	-	+	-	+
10.	Carbohydrates	-	-	-	+
11.	Proteins	+	-	-	+
12.	Steroids and sterols	-	-	+	+
13.	Fixed oils	-	+	-	-

This study has revealed the presence of phytochemicals that were considered as active medicinal biochemical constituents. The extracts of Bauhinia purpurea when tested with different solvents of varying polarity index showed positive results for important phytochemicals such as alkaloids, anthocyanin, triterpenoids, theobromine, phenols, tannins, carbohydrates, flavanoids, saponin and glycosides. Among the different solvents used the maximum results were obtained from the aqueous extract of the plant.

After preliminary phytochemical screening, the extracts were subjected to instrumental analysis for characterization. In this study three predominant characterization methods were adopted namely TLC, HPLC and GC-MS.

Thin layer chromatography:

TLC is performed to separate the non-volatile compounds in the mixture. There are 3 basic steps in TLC namely spotting, development, and visualization. Representative silica TLC separations of Bauhinia purpurea plant extract using chloroform and ethyl acetate were shown in Fig 2. Separation was done using three solvent systems namely (I) Chloroform (II) Ethyl acetate: n- hexane (6:4), (III) n-hexane: Ethyl acetate: methanol: chloroform (4:4:2:1). Separation of chloroform extract using solvent system (I) and ethyl acetate extract using solvent system (III) resulted in 4 bands whereas 5 bands were resolved for chloroform extract using solvent system (II) and (III). These results demonstrates that different numbers of bands were revealed when different solvent systems used and there were minimal overlaps indicating that choice of solvent system and extracts can affect the number of compounds separated on a chromatogram.

From the above table 3 it is inferred that only chloroform and ethyl acetate extracts were resolved into bands in TLC plates whereas ethanolic and aqueous extract of the plant showed no separation.

Table 3 Retention factor for each plant extract in three different solvent systems

Extracts	Rf value of solvent system (I)	Rf value of solvent system (II)	Rf value of solvent system (III)
Chloroform	0.08,0.2, 0.48, 0.5	0.05, 0.4, 0.5, 0.54, 0.6	0.27, 0.35, 0.54, 0.6, 0.65
Ethyl acetate	NS	NS	0.2, 0.3, 0.4, 0.6
Ethanol	NS	NS	NS
Water	NS	NS	NS

(NS- No separation)

Screening of antioxidant activity by Bioautography:

The TLC plates with the separated compounds are used for bioassay where in DPPH was sprayed on to the plates. The presence of antioxidant activity was noticed in the plate with chloroform extract where there was a change in colour and this colour change was not significant in other plates with other extracts. The presence of antioxidants was observed in Fig 2 - A due to change of DPPH spray from purplish colour to yellowish spot on the bands of TLC plate. In the Fig 2 - A the marked portion represents the presence of antioxidants exactly in the second band (from the top) of chloroform extract in solvent system (I) having the retention factor 0.48 which confirms that this group of antioxidants maybe non- polar in nature.

Fig 2 TLC analysis of phytochemicals

A-TLC bioassay for antioxidant activity; I Chloroform extract- Solvent system 1; II Chloroform extract -solvent system II; III- Chloroform extract -solvent system III; EA-I Ethyl acetate extract- Solvent system 1

Antioxidant activity of the plant extract:

The TLC bioassay reported the antioxidant activity in the chloroform extract and hence this extract (in different concentrations) was used to quantify the scavenging activity. Ascorbic acid was used as standard and the experiments were carried out in triplicates (Table 4).

Table 4 Antioxidant activity of the plant extract

Concentrations of plant extract used (mg/ml)	Scavenging activity (%)
Concentrations of plant extract used (mg/ml)	Control	Plant extract
0.1	69.21±0.62	66.76±0.45
0.5	73.45±0.35	75.03±0.01
0.75	82.22±0.38	83.33±0.34
1	85.96±0.32	84.02±0.48

The result clearly indicates the effect of concentration of the plant extract on the scavenging activity. As the concentration increases the activity was found to increase and the values were promising even in low concentration. The activity of the plant extract was on par with that of the standard making it a promising source of antioxidant compounds. Out of the varied group of plant metabolites, polyphenols are one category that exhibits a range of biological activities that include antiinflammatory, antiallergic, antibacterial, hepatoprotective, antithrombotic, antiviral, antioxidant, anticarcinogenic and cardioprotective activities¹⁶. In fact antioxidant activity of the plant is directly correlated with the phenolic content^17,18. The flavonoids, a type of polyphenol are known to possess significant antioxidant activities. This is due to the hydroxyl groups in their structure that prevents oxidative damage caused by the free radicals^19,20. In our study preliminary phytochemical analysis showed the presence of flavonoids that may be responsible for the antioxidant activity.

Anti-bacterial activity against Pseudomonas aeruginosa strain:

Among the different solvent extracts studied ethanolic extract showed notable antibacterial activity against Pseudomonas aeruginosa strain compared to all other extracts. Different concentrations of the plant extract were used and it was observed that as the concentration increased there is an increase in the activity. Good inhibition zone was obtained with a concentration of 60µl (8 mm) and 80µl (10 mm) of ethanolic extracts respectively which is on par with the standard used (Fig 3). It was observed that extracts prepared in organic solvents consistently displayed better antimicrobial activity than that of aqueous extracts which may be due to the ability of these active compounds to be highly soluble in organic solvents.

Fig 3 Activity of methanolic extract against Pseudomonas aeruginosa

This activity observed may be due to the presence of alkaloids present in the ethanolic extract, as alkaloids are the class of phytochemicals that are known to possess antimicrobial activity. It was postulated that this acts by intercalating with the DNA of parasites, thereby resulting in the destruction of entire organism²¹. A study earlier, by Negi et al¹⁴ demonstrated the effectiveness of methanol extract of Bauhinia pupurea leaves as inhibitors of microbes compared to the aqueous extracts. Also, flavonoids are another class of secondary metabolites in plants that exhibit antimicrobial activity through formation of a complex with the bacterial cell wall^15,22. The presence of alkaloids and flavonoids in the biochemical tests substantiates the effectiveness of plant extract as antimicrobial agent.

HPLC analysis:

The plant extract was subjected to HPLC analysis to understand the possible compounds present based on the peaks that is obtained from HPLC (Fig 4). The results obtained from HPLC crude extract profile for Bauhinia purpurea showed few major peaks at the retention time 2.336 min, 2.554 min, 2.842 min and 4.127 min. The peaks obtained indicate the presence of different compounds at different retention times as HPLC was carried out for the crude extract only that would be identified after purification. But for the identification of the specific compounds, individual bands have to be subjected to HPLC. Moreover, processing of a crude sample with the right kind of solvent for sample reconstitution is known to have significant relevance on the overall success of natural product isolation.

Fig 4 HPLC result of the plant extract

Fig 5 GC MS Chromatogram of the plant extract

Table 5 GC-MS profile of plant extract

Sno	Retention time	Phytocompounds name	Molecular formula	MW
1	3.439	Silanediol, dimethyl	C₂H₈O₂Si	92.029356
2	4.033	Cyclotrisiloxane, hexamethyl	C₆H₁₈O₃Si₃
3	5.504	Benzeneethanamine, N-[(4-hydroxy)hydrocinnamoyl]	C₁₇H₁₉NO₂	269.141579
4	5.630	Ethyl(dimethyl)benzyloxysilane	C₁₁H₁₈OSi	194.112692
5	7.406	Acetic acid, N'-[3-(1-hydroxy-1-phenylethyl)phenyl]hydrazide	C₁₆H₁₈N₂O₂	270.136827
6	8.409	Cyclotetrasiloxane, octamethyl	C₈H₂₄O₄Si₄	296.075165
7	10.028	6,6,8,8,10,10-Hexamethyl-2,5,7,9,11,14-hexaoxa-6,8,10-trisilapentadecane	C₁₂H₃₂O₆Si₃	356.15067
8	11.871	Benzaldehyde, 2,5-bis[(trimethylsilyl)oxy]	C₁₃H₂₂O₃Si₂	282.110748
9	12.992	Cyclopentasiloxane, decamethyl	C₁₀H₃₀O₅Si₅	370.093956
10	15.221	1,3-Dioxane, 4-(hexadecyloxy)-2-pentadecyl	C₃₅H₇₀O₃	538.532497
11	15.786	Acetic acid, [bis[(trimethylsilyl)oxy]phosphinyl]-, trimethylsilyl ester	C₁₁H₂₉O₅PSi₃	356.106041
12	17.806	Cyclohexasiloxane, dodecamethyl	C₁₂H₃₆O₆Si₆	444.112747
13	22.219	Cycloheptasiloxane, tetradecamethyl	C₁₄H₄₂O₇Si₇	518.13154
14	24.953	Diethyl Phthalate	C₁₂H₁₄O₄	222.089209
15	26.186	Cyclooctasiloxane, hexadecamethyl	C₁₆H₄₈O₈Si₈	592.15033
16	29.618	Cyclononasiloxane, octadecamethyl	C₁₈H₅₄O₉Si₉	666.16912
17	31.735	Hexadecanoic acid, methyl ester (Palmitic acid)	C₁₆H₃₂O₂	256.43
18	32.701	Cyclodecasiloxane, eicosamethyl	C₂₀H₆₀O₁0Si₁₀	740.187912
19	35.019	9,12-Octadecadienoic acid, methyl ester, (E,E) (Methyl linolelaidate)	C₁₉H₃₄O₂	294.4721
20	39.595	Ricinoleic acid	C₁₈H₃₄O₃	298.461

GC-MS analysis:

The GC-MS analysis of plant extract was carried out (Fig 5) and the list of compounds identified are tabulated below (Table 5).

The GC-MS analysis of the plant extract showed the presence of many compounds at different retention times. The most significant compounds were identified to be Hexadecanoic acid, methyl ester; 9,12-Octadecadienoic acid, methyl ester and Ricinoleic acid that is reported to have antioxidant, analgesic and anti-inflammatory activity at the retention time 31.735, 35.019 and 39.595 respectively. Further studies on purifying the crude extract and separation of compounds would provide an insight into the beneficial compounds of the plant.

Isolation and preliminary identification of endophytic bacteria:

A total of three endophytic bacteria were isolated from Bauhinia purpurea and named as Bb1, Bb2 and Bb3 (Fig 6).

Fig 6 Isolation of endophytic bacteria from the leaf segments and stem of Bauhinia purpurea.

The isolated endophytes were initially identified by standard biochemical tests and the results are provided in table 6.

Table 6 Biochemical test results for the endophytic bacteria

Test name	Results
Test name	Bb1	Bb2	Bb3
Gram staining	Gram +, rod	Gram -, cocci	Gram +, rod
Motility	+	+	-
Single enzyme test
Catalase	+	+	+
Indole	-	-	-
Urease	-	-	+
Assays for metabolic pathways
Methyl red	-	+	+
Voges Proskauer	-	+	+
Hydrogen production	-	-	-
Single substrate utilization test
Acetate	-	-	-
Citrate	-	-	-
Starch	+	-	-

(‘+’ represents positive and ‘-’ represents negative)

After the preliminary characterization, 16srRNA sequencing was carried out for the endophytic bacteria. The best use of 16s rRNA gene sequence is to provide genus and species identification for isolates that do not fit any recognized biochemical profiles, for strains generating only a “low likelihood” or “acceptable” identification according to commercial systems, or for taxa that are rarely associated with human infectious diseases. The ability of defining the phylogenetic relationships of bacteria and indeed all life-forms is dependent on comparing a stable part of genetic code that include genes that code for 5S, 16S and the 23S rRNA and the spaces between these genes with 16S being the most common for taxonomic purposes especially for bacteria^23-27. The prime reasons for 16S rRNA being the most predominantly used technique are i) its presence in almost all bacteria, often existing as a multigene family, or operons; (ii) the function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of time (evolution); and (iii) the 16S rRNA gene (1,500 bp) is large enough for informatics purposes^28.

The 16SrRNA sequencing was carried out by initially extracting the DNA followed by PCR amplification of the 16s rDNA gene. The amplified products are sequenced and the sequence obtained is compared with the database sequence using BLAST program. The results for Bb1 and Bb2 were not substantial as the sequencing was not significantly successful with universal primers. Hence studies to identify the strain are going on with the help of specific primers.

The experiment has been successful for the isolate Bb3 and the results are provided in table 7. From the results it can be inferred that the isolate belongs to Bacillus sp as it is evident from the biochemical tests earlier. The isolate showed 82% similarity with other strains of Bacillus sp. The phylogenetic tree (Fig 7) with the matching sequences also revealed that the isolate Bb3 was closely associated with other strains of Bacillus sp. Genera level identification was possible as the similarity was not very high and further confirmation is required to validate the strain at the species level.

Table 7 BLAST result for the isolate Bb3

Description	Max score	Total score	Query cover	E value	Ident	Accession
Bacillus oleronius strain 30N2-10 16S ribosomal RNA gene, partial sequence	466	466	53%	5e-127	82%	JN366727.1
Uncultured bacterium clone CU18 16S ribosomal RNA gene, partial sequence	464	464	53%	2e-126	82%	KC414644.1
Bacillus sp. strain JST17 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KX397511.1
Bacillus oleronius strain PD14 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KY773590.1
Bacillus oleronius strain PD3 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KY773585.1
Bacillus sp. strain Hc010 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KY465508.1
Bacterium strain PGLB7 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KX131099.1
Bacillus oleronius strain M1/25 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KX826966.1
Bacillus sp. CSB17 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KX289458.1
Uncultured Bacillus sp. clone UOC/PTS/BAC/2F2 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KU531728.1
Bacillus oleronius strain DMB29 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KT274774.1
Bacillus oleronius strain DMB28 16S ribosomal RNA gene, partial sequence	462	462	53%	6e-126	82%	KT274773.1

Fig 7 Phylogenetic relationship between the isolate Bb3 and its matching sequences.

Antibacterial activity of the endophytic bacteria:

The ability of the endophytic bacterial isolates to inhibit the growth of Pseudomonas aeruginosa was evaluated using agar well diffusion assay along with standard Streptomycin. The zones of inhibition recorded are Streptomycin (Control-15mm), Bb1 (17mm), Bb2 (14 mm) and Bb3 (18 mm). These results clearly indicate the potential of these bacteria to inhibit the growth of the pathogen as the zones were on par with the standard antibiotic used. Further the antibacterial potential of these bacteria would be extended to other pathogens also.

Antioxidant activity of the endophytic bacteria:

Using ascorbic acid as control, the supernatant of the three endophytic bacteria was used to check the ability of bacteria to scavenge the free radicals. The three bacteria showed good amount of anti-oxidant activity based on the % scavenging activity. The activity was found to be 79.3±0.57 by isolate Bb1, 82.7±0.39 by isolate Bb2 and 84.3±0.27 by isolate Bb3 while the control showed 85.5±0.57 scavenging activity. The values obtained are on par with the control hence suggesting the ability of these endophytes to produce antioxidant compounds. This is a preliminary report on the potential of these endophytic bacteria to produce useful secondary metabolites.

CONCLUSION:

In the present study one of the most important medicinal plants B.purpurea was selected and the phytocompounds present in it were screened by standard biochemical tests followed by instrumental analysis. The extract of plant showed appreciable antioxidant and antimicrobial activity due to the presence of alkaloids and flavonoids. From the GC-MS result three compounds namely Hexadecanoic acid, methyl ester, 9, 12-Octadecadienoic acid, methyl ester and Ricinoleic acid were present. These compounds were reported to have antioxidant and anti-inflammatory activity. The ethanolic extract of the plant conferred decent antimicrobial activity against the most important pathogen Pseudomonas aeruginosa, while the chloroform extract showed good antioxidant activity. Further an attempt has been made to isolate endophytic bacteria from Bauhinia purpurea. Three endophytic bacteria were isolated (bb1, Bb2, Bb3) and Bb3 was identified as a strain that belongs to Bacillus sp. The three endophytic bacteria were reported to show notable antibacterial activity against Pseudomonas aeruginosa and significant antioxidant activity. Further studies are in progress with respect to endophytes and their biological activities.

ACKNOWLEDGEMENT:

The authors are thankful to Sathyabama Institute of Science and Technology for giving us the opputunity to carry out the work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 04.01.2018 Modified on 08.02.2018

Research J. Pharm. and Tech 2018; 11(5):1867-1876.

DOI: 10.5958/0974-360X.2018.00347.5

Parts used	Method of extraction	Solvent	Yield (%)	Colour and consistency	Fluorescence analysis
Parts used	Method of extraction	Solvent	Yield (%)	Colour and consistency	Under Visible light	Under UV light
Leaf, stem and flower mixture	Serial exhaustive method	Chloroform	6.2	Dark greenish gum	Green	Pink
		EA	9	Greenish sludge	Dark green	Pink
		Ethanol	10.4	Light greenish mesh	Light green	Pink
		Water	8.5	Brownish sludge	Tawny brown	Green